17β-Estradiol Modulates Cancer Cell-Fibroblast Communication via Autophagy-Mediated Extracellular Vesicle Secretion and Promotes Poor Prognosis in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Smoking is the primary etiological factor, but growing evidence suggests the involvement of estrogen in its development and progression, although its role remains unclear. This study explores: (i) the effects induced by estrogen, namely, 17β-estradiol (E2), alone or in combination with a mixture of inflammatory cytokines (Mix), in two human NSCLC cell lines, A549 and Calu1, and (ii) whether and how tumor cells can modulate the activation of normal lung fibroblasts. We found that E2 significantly enhances migration, invasion, and epithelial-mesenchymal transition in NSCLC cells, as well as their resistance to cisplatin, particularly in combination with Mix. Pharmacological inhibition of ERβ reversed the E2-induced effects, implicating ERβ in E2-mediated signaling. Furthermore, E2 increased autophagic flux and induced a shift toward secretory autophagy and the release of extracellular vesicles, which activated normal lung fibroblasts, as demonstrated by the increased expression of α-SMA, FAP, PDGFR-β, and PDPN. The clinical relevance of these data was supported by computational analyses revealing an elevated expression of the Mix gene signature, including TGF-β, IL-6, IL-8, CCXL-16, and ERβ, which was associated with shorter overall survival in NSCLC patients. This molecular profile was linked to the elevated expression of secretory autophagy genes and cancer-associated fibroblast markers. Validation in three large clinical cohorts (TCGA-LUNG, OAK and POPLAR) strengthens the clinical relevance of this E2-related pro-tumor axis while suggesting a promising therapeutic avenue for NSCLC patients.